Expression Of PARP Research Articles

Dehydrocostus lactone inhibits the proliferation and metastasis of hepatocellular carcinoma cells via modulating p53-p21-CDK2 signaling pathway

BackgroundHepatocellular carcinoma (HCC), a malignancy with high mortality and recurrence rates, has very limited options for treatment strategies. Dehydrocostus lactone (DL) is the principal quality marker extracted from Aucklandia lappa Decne. and has been demonstrated to possess excellent anticancer activity. The key purpose of this study was to explore the therapeutic effects and potential mechanisms of DL on HCC. MethodsThe effect of DL on the cell viability of HCC cell lines (HepG2 and SK-HEP-1) and normal human hepatocyte cell line (L-O2) was examined by CCK8, colony formation, and BeyoClickTMEdU-488 assays; Hoechst 33,258 staining and flow cytometry were utilized to determine the impact of DL on apoptosis; the sensitivity of HCC cells to DL was explored by adding Z-VAD-fmk. In addition, the expression of apoptosis-related proteins Bax, Bcl-2, and PARP was detected by western blotting; the expression of p-H2AX was investigated by western blotting and immunofluorescence staining; cell cycle distribution was observed using flow cytometry; the migration ability and invasiveness of HCC cells were assessed by wound healing assay and transwell assay; immunoblotting was applied to visualize the levels of EMT markers in the two HCC cell lines. Transcriptome sequencing was performed to reveal the underlying mechanisms of DL anti-tumor; western blotting and qRT-PCR were employed to verify the mRNA and protein abundance of p53, p21, and CDK2; HepG2 xenograft in mice was used to test the anti-tumor effects of DL in vivo. ResultsDL suppressed the proliferation of HCC cell lines (HepG2 and SK-HEP-1) but had little effect on a normal human liver cell line (L-O2). In addition, DL also induced apoptotic death of the HCC cells by activating Bax and downregulating Bcl-2. The antiproliferative effects of DL could be attributed to increased DNA damage and G1-phase cell cycle arrest. Moreover, DL inhibited HCC cell invasiveness and migration in vitro by decreasing the levels of β-catenin, N-cadherin, and TCF8/ZEB1, and increasing the E-cadherin level. RNA sequencing indicated that DL exerted its anti-hepatoma effects partly via regulation of p53-p21-CDK2 signaling. These results were validated by our in vivo experiments, in which DL markedly suppressed the growth of HepG2 xenografts in a mouse model without any toxic side effects, which corresponded to decreased expression of Ki67 and MMP9 in the tumor tissues. ConclusionDL has significant anti-cancer effects in both in vivo and in vitro HCC models, and therefore could be further developed as a promising drug for the treatment of HCC.

Preclinical evaluation of oncolytic potential human rotavirus Wt 1-5 in gastric adenocarcinoma.

Despite advances in biomedical research, gastric cancer remains the leading cause of morbidity and mortality worldwide due to the limited efficacy of conventional therapies. In recent decades, oncolytic viruses have emerged as a biological therapeutic alternative to cancer due to their selectivity, effectiveness, and low toxicity. However, clinical trials have shown that developing a virus with selectivity for multiple tumor receptors and the ability to penetrate and diffuse through the tumor microenvironment to reactivate the immune system remains challenging. This study aimed to examine the oncolytic potential of tumor cell-adapted rotavirus Wt1-5 in gastric adenocarcinoma samples. This study focused on determining the propagation capacity of the RV Wt1-5 through the tumor and the importance of the expression of cell surface co-receptors, including integrin β3, protein disulfide isomerase (PDI), and heat shock proteins (Hsp-90, -70, -60, -40, and Hsc 70), during infection of tumor cells. These proteins were found to be differentially expressed in tumor cells compared to adjacent non-tumor cells. Preincubation of gastric tumor cells with antibodies against these proteins decreased rotavirus infections, validating their importance in the binding and entry of RV Wt1-5 into tumor cells, as previously reported. Upon RV infection, apoptosis was one of the types of death that was observed. This was evidenced by evaluating the expression of CASP-3, -9, PARP, cytochrome C, Bax, Bid, p53, and Bcl-2, as well as observing morphological changes such as chromatin margination, nuclear condensation, and fragmentation. Finally, at 60 h.p.i, histological analysis revealed that oncolysis compromised the entire thickness of the tumor. Therefore, the results suggest that RV Wt1-5 could be a novel therapeutic agent co-adjuvant agent for conventional and targeted therapies in managing GC. Ex vivo infection of the tumor tissue model showed characteristics of an immune response that could be explored in future studies.

Curcumol metabolized by rat liver S9 fraction and orally administered in mouse suppressed the proliferation of colon cancer in vitro and in vivo.

Following 3R (reduction, refinement, and replacement) principles, we employed the rat liver S9 fraction to mimic liver metabolism of curcumol having high in vitro IC50 on cancer cells. In HCT116 and HT29 colon cancer cells, the metabolites of curcumol by S9 fraction exerted more enhanced activity in inducing cell cycle arrest and apoptosis via regulating the expression of cyclin D1, CDK1, p21, PARP and Bcl-2 than curcumol. In addition, oral administration of curcumol at 4mg/kg BW significantly suppressed the development of colon tumor induced by azoxymethane/dextran sulfate sodium, and induced cell cycle arrest and apoptosis in tumor tissues. In mass analysis, curcumenol and curzerene were identified as the metabolites of curcumol by S9 fraction metabolism. Taken together, curcumol metabolites showed the enhanced suppressive effect on colon cancer, suggesting that S9 fraction can be considered as simple, fast, and bio-mimicking platform for the screening of chemical libraries on different chronic diseases.

PET imaging of PARP expression using 68Ga-labelled inhibitors

PurposeImaging the PARP expression using 18F probes has been approved in clinical trials. Nevertheless, hepatobiliary clearance of both 18F probes hindered their application in monitoring abdominal lesions. Our novel 68Ga-labelled probes aim for fewer abdominal signals while ensuring PARP targeting by optimizing the pharmacokinetic properties of radioactive probes.MethodsThree radioactive probes targeted PARP were designed, synthesized, and evaluated based on the PARP inhibitor Olaparib. These 68Ga-labelled radiotracers were assessed in vitro and in vivo.ResultsPrecursors that did not lose binding affinity for PARP were designed, synthesized, and then labelled with 68Ga in high radiochemical purity (> 97%). The 68Ga-labelled radiotracers were stable. Due to the increased expression of PARP-1 in SK-OV-3 cells, the uptake of the three radiotracers by SK-OV-3 cells was significantly greater than that by A549 cells. PET/CT imaging of the SK-OV-3 models indicated that the tumor uptake of 68Ga-DOTA-Olaparib (0.5 h: 2.83 ± 0.55%ID/g; 1 h: 2.37 ± 0.64%ID/g) was significantly higher than that of the other 68Ga-labelled radiotracers. There was a significant difference in the T/M (tumor-to-muscle) ratios between the unblocked and blocked groups as calculated from the PET/CT images (4.07 ± 1.01 vs. 1.79 ± 0.45, P = 0.0238 < 0.05). Tumor autoradiography revealed high accumulation in tumor tissues, further confirming the above data. PARP-1 expression in the tumor was confirmed by immunochemistry.ConclusionAs the first 68Ga-labelled PARP inhibitor, 68Ga-DOTA-Olaparib displayed high stability and quick PARP imaging in a tumor model. This compound is thus a promising imaging agent that can be used in a personalized PARP inhibitor treatment regimen.

Evaluation of Potential Neuroprotective Effects of Vanillin Against MPP+/MPTP-Induced Dysregulation of Dopaminergic Regulatory Mechanisms in SH-SY5Y Cells and a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative condition. The pathogenesis of PD is still unknown, and drugs available for PD treatment either have side effects or have suboptimal efficacy. Flavonoids are potent antioxidants having little toxicity with extended use, suggesting they might hold promising therapeutic potential against PD. Vanillin (Van) is a phenolic compound that has exhibited neuroprotective properties in various neurological disorders, including PD. However, the neuroprotective role of Van in PD and its underlying mechanisms are scarce and therefore need more exploration. Here, we evaluated the neuroprotective potential of Van and its associated mechanisms against MPP+/MPTP-induced neuronal loss in differentiated human neuroblastoma (SH-SY5Y) cells and the mouse model of PD. In the present study, Van treatment significantly enhanced the cell viability and alleviated oxidative stress, mitochondrial membrane potential, and apoptosis in MPP+-intoxicated SH-SY5Y cells. Moreover, Van significantly ameliorated the MPP+-induced dysregulations in protein expression of tyrosine hydroxylase (TH) and mRNA expressions of GSK-3β, PARP1, p53, Bcl-2, Bax, and Caspase-3 genes in SH-SY5Y cells. Similar to our in vitro results, Van significantly alleviated MPTP-induced neurobehavioral dysregulations, oxidative stress, aberrant TH protein expressions, and immunoreactivity in SNpc of mice brains. Treatment of Van also prevented MPTP-mediated loss of TH-positive intrinsic dopaminergic neurons to SNpc and TH-fibers projecting to the striatum of mice. Thus, Van exhibited promising neuroprotective properties in the current study against MPP+/MPTP-intoxicated SH-SY5Y cells and mice, indicating its potential therapeutic properties against PD pathology.

NAMPT and PARylation Are Involved in the Pathogenesis of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of very high prevalence, especially in childhood, with no specific treatment or cure. As its pathogenesis is complex, multifactorial and not fully understood, further research is needed to increase knowledge and develop new targeted therapies. We have recently demonstrated the critical role of NAD+ and poly (ADP-ribose) (PAR) metabolism in oxidative stress and skin inflammation. Specifically, we found that hyperactivation of PARP1 in response to DNA damage induced by reactive oxygen species, and fueled by NAMPT-derived NAD+, mediated inflammation through parthanatos cell death in zebrafish and human organotypic 3D skin models of psoriasis. Furthermore, the aberrant induction of NAMPT and PARP activity was observed in the lesional skin of psoriasis patients, supporting the role of these signaling pathways in psoriasis and pointing to NAMPT and PARP1 as potential novel therapeutic targets in treating skin inflammatory disorders. In the present work, we report, for the first time, altered NAD+ and PAR metabolism in the skin of AD patients and a strong correlation between NAMPT and PARP1 expression and the lesional status of AD. Furthermore, using a human 3D organotypic skin model of AD, we demonstrate that the pharmacological inhibition of NAMPT and PARP reduces pathology-associated biomarkers. These results help to understand the complexity of AD and reveal new potential treatments for AD patients.

Combination of Everolimus and Bortezomib Inhibits the Growth and Metastasis of Bone and Soft Tissue Sarcomas via JNK/p38/ERK MAPK and AKT Pathways.

The combination of the mammalian target of rapamycin and proteasome inhibitors is a new treatment strategy for various tumors. Herein, we investigated the synergistic effect of everolimus and bortezomib on tumor growth and metastasis in bone and soft tissue sarcomas. The antitumor effects of everolimus and bortezomib were assessed in a human fibrosarcoma (FS) cell line (HT1080) and mouse osteosarcoma (OS) cell line (LM8) by MTS assays and Western blotting. The effects of everolimus and bortezomib on HT1080 and LM8 tumor growth in xenograft mouse models were evaluated using tumor volume and the number of metastatic nodes of the resected lungs. Immunohistochemistry was used to evaluate cleaved PARP expression. The combination therapy decreased FS and OS cell proliferation compared with either drug alone. This combination induced more intense p-p38, p-JNK, and p-ERK and activated apoptosis signals, such as caspase-3, compared with single-agent treatment. The combination treatment reduced p-AKT and MYC expression, decreased FS and OS tumor volumes, and suppressed lung metastases of OS. The combination therapy inhibited tumor growth in FS and OS and metastatic progression of OS via the JNK/p38/ERK MAPK and AKT pathways. These results could aid in the development of new therapeutic strategies for sarcomas.

Exploring the Control of PARP1 Levels in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a leading cause of mortality from gynecologic malignancies worldwide. Although a transformative improvement has been shown with the introduction of PARP (poly(ADP-ribose) polymerase) inhibitors, the emergence of resistance to these drugs represents a therapeutic challenge. Hence, expanding our understanding of mechanisms behind the control of PARP1 expression can provide strategic guidance for the translation of novel therapeutic strategies. The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors critically involved in the regulation of important cellular functions. Notably, we recently demonstrated that, in cervical cancer cells, STAT1 controls PARP1 levels through multiple mechanisms, possibly involving also STAT3. Here, we tested the hypothesis that a similar mechanism might be operative in HGSOC. To this end, the impact of STAT1/STAT3 modulation on PARP1 expression was assessed in established and primary HGSOC cells, and molecular biology studies proved that STAT1 might act at both transcriptional and post-transcriptional levels to modulate the PARP1 level. Notably, bioinformatics analysis of TCGA databases demonstrated that increased STAT1 mRNA expression levels are associated with a favorable prognosis and with response to chemotherapy in HGSOC patients. Our findings suggest an alternative strategy for targeting HGSOC cells based on their dependency on PARP1.

A Novel Derivative of Curcumol, HCL-23, Inhibits the Malignant Phenotype of Triple-Negative Breast Cancer and Induces Apoptosis and HO-1-Dependent Ferroptosis.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype of breast cancer. Curcumol, as a natural small molecule compound, has potential anti-breast cancer activity. In this study, we chemically synthesized a derivative of curcumol, named HCL-23, by structural modification and explored its effect on and underlying mechanism regarding TNBC progression. MTT and colony formation assays demonstrated that HCL-23 significantly inhibited TNBC cells proliferation. HCL-23 induced G2/M phase cell cycle arrest and repressed the capability of migration, invasion, and adhesion in MDA-MB-231 cells. RNA-seq results identified 990 differentially expressed genes including 366 upregulated and 624 downregulated genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) revealed that these differentially expressed genes were obviously enriched in adhesion, cell migration, apoptosis, and ferroptosis. Furthermore, HCL-23 induced apoptosis via the loss of mitochondrial membrane potential and the activation of the caspase family in TNBC cells. In addition, HCL-23 was verified to trigger ferroptosis through increasing cellular reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation levels. Mechanistically, HCL-23 markedly upregulated the expression of heme oxygenase 1 (HO-1), and the knockdown of HO-1 could attenuate ferroptosis induced by HCL-23. In animal experiments, we found that HCL-23 inhibited tumor growth and weight. Consistently, the upregulation of Cleaved Caspase-3, Cleaved PARP, and HO-1 expression was also observed in tumor tissues treated with HCL-23. In summary, the above results suggest that HCL-23 can promote cell death through activating caspases-mediated apoptosis and HO-1-dependent ferroptosis in TNBC. Therefore, our findings provide a new potential agent against TNBC.

Abstract 2409: A synergistic effect of miR-21-5p inhibitor and PARP1 inhibitor to enhance the radiosensitivity of oral squamous cell carcinoma

Abstract Radiotherapy is one of the standard treatments for oral squamous cell carcinoma (OSCC). Radiotherapy damages cancer cells mainly by inducing DNA double-strand breaks, but some advanced OSCC can effectively repair DNA damages, resulting in radiotherapy resistance. TCGA data showed that miR-21-5p was highly expressed in advanced OSCC and correlated with prognosis. However, the involvement of miR-21-5p in OSCC’s response to radiotherapy and the underlying mechanism are still unclear. When treated with a miR-21-5p inhibitor, we found OSCC cell lines (SAS and CAL33) became more sensitive to X-ray radiation at 2Gy (the commonly used single-dose in conventional radiotherapy), which was supported by the evidence that more resident 53BP1-marked-foci for non-repaired DNA double strand breaks (1.61±0.54 folder more in SAS, 1.47±0.49 folder more in CAL33, n=4, p&amp;lt;0.05) and less colony-forming capacity (14.62±2.36% in SAS, 18.65±4.32% in CAL33, n=3, p&amp;lt;0.05) than untreated control cells were observed at 24hr after radiation. However, the expression of PARP1, a DNA damage sensor for recognizing DNA damage and facilitating DNA repair, was significantly increased (1.38±0.12, n=4, p&amp;lt;0.05) in SAS cells treated with miR-21-5p inhibitor. In order to achieve a better tumor control, we tested the combined treatments with miR-21-5p inhibitor and PARP1 inhibitor (Olaparib) on SAS cells, which resulted in a synergistic effect in inhibition of the repair of DNA double strand breaks. The residual 53BP1 foci after 2Gy X-ray irradiation was increased 1.45±0.06 folder more when inhibiting both miR-21-5p and PARP1, while the colony-forming capacity of irradiated SAS cells was decreased by 39.55±5.89% (p&amp;lt;0.0001) in the same conditions. This work provided novel insights on the molecular mechanism of miR-21-5p regulated cell response to radiotherapy, which may give rise to potential therapeutic strategy for treatment of advanced OSCC. Citation Format: Lin Ma, Weitao Hu, Rui Fu, Anhui Wu, Qiangyong Deng, Qi Liu. A synergistic effect of miR-21-5p inhibitor and PARP1 inhibitor to enhance the radiosensitivity of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2409.

Abstract 2774: Moesin-induced adriamycin resistance through IL-6/MSN/STAT3 axis and therapeutic efficacy of atovaquone in chemotherapy resistant triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high frequency of chemotherapy resistance. Moesin (MSN) is a cytoskeleton linker discovered as a novel oncogene in our previous proteomics analysis. Here, we evaluated the molecular mechanisms of MSN and how it functions in response to chemotherapy, which remains unclear in TNBC. Method: The 178 in-house TNBC patient cohort and METABRIC data set were enrolled for clinicopathologic analysis. The multiple TNBC four-cell lines were used to establish MSN knock-down and over-expressed in vitro models using a lentiviral system. RNA sequencing was analyzed with MDA-MB-231 parent cell and MDA-MB-231/ADR12 (adriamycin resistance cell line). Invasion and migration assay was done. The drug response based on MSN gene expression was observed by cell viability assay, western blot, and AV/PI flow cytometry. Drug resistance concerning cancer stemness was confirmed with sphere formation and ALDH activity assay. Immunofluorescence and immunoprecipitation were performed. The combination index(CI), which is to define the synergism of combined treatment was calculated with the Compusyn program. Results: The cohort with METABRIC and our in-house clinical data showed concordant findings that overexpressed MSN mRNA and protein expressions have a poor prognosis in TNBC patients. The MSN protein expression in TNBC was increased compared to the protein level in both the normal human tissues and non-TNBC cell lines. Overexpressed MSN induced high cell proliferation activity, invasion, and migration ability. The apoptosis rate (AV+/PI+) along with cleaved PARP and caspase-3 expression was increased in TNBC cell lines with MSN downregulation. Further tests to determine the role of MSN in adriamycin resistance showed an increased number and size of spheres in the MSN overexpressed cell lines. Moreover, the protein and mRNA levels of MSN expression in MDA-MB-231/ADR12 were significantly higher than the level in the matched parental cell line. MSN and phospho-STAT3(T705) were increased by treatment of IL-6 in TNBC cell lines. The direct binding between MSN and STAT3, and the co-localization of MSN and pSTAT3 were consistently confirmed by co-immunoprecipitation and immunofluorescence, which were all activated by IL-6. Blocking the STAT3 pathway with atovaquone restored ADR response in MSN overexpressed tumor cells. CI value on the combined treatment of ADR and atovaquone in the MDA-MB-231/ADR12 cell line showed higher synergetic efficacy than the parental cell line. Conclusion: This research demonstrates the involvement of the MSN in doxorubicin resistance through IL-6/MSN/STAT3 pathway and provides a novel therapeutic candidate for TNBC patients. Citation Format: Cheng Hyun Lee, Da Sol Kim, Soo Young Park, Han Suk Ryu. Moesin-induced adriamycin resistance through IL-6/MSN/STAT3 axis and therapeutic efficacy of atovaquone in chemotherapy resistant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2774.

Abstract 2832: Plk1 signaling as a therapeutic target for HPV- head and neck cancer

Abstract Current frontline chemotherapy and radiotherapy for head and neck cancer (HNC) is insufficient as ~60% of patients relapse within two years, indicating a need to identify novel targeted therapies to improve survival outcomes for HNC patients. Human papilloma virus (HPV) incidence is a major cause of head and neck squamous cell carcinoma (HNSCC) in addition to tobacco use. HPV- HNSCC patients are less responsive to frontline treatment than HPV+ HNSCC patients. However, HPV- and HPV+ HNSCC patients are treated similarly. The cell cycle serine-threonine kinase polo-like kinase 1 (Plk1) is known to be overexpressed in HNC. The Cancer Genome Atlas (TCGA) revealed that high expression of Plk1 correlates with worse survival in HNSCC patients (p&amp;lt;0.05). Yet, Plk1 is expressed at similar levels when comparing HPV- and HPV+ HNSCC patients. Therefore, in this study we sought to establish if Plk1 inhibition with onvansertib (currently in Phase I/II clinical trials) alone and in combination with radiation would inhibit HPV- and HPV+ HNSCC growth. We used two HPV+ (UMSCC47, UDSCC2), and two HPV- (HN5, Cal27) human HNSCC cell lines. First, we confirmed that Plk1 inhibition with onvansertib stalls HNSCC cells at the G2/M phase of the cell cycle (n=3). Then, we determined that HNSCC cell viability is reduced in a dose-dependent manner after Plk1 inhibition with onvansertib. Surprisingly, HPV- HNSCC cells exhibited a 2-fold decrease in cell viability as compared to HPV+ HNSCC cells (n=3). These findings were confirmed when assessing 3D spheroid growth of HNSCC cells. Plk1 inhibition with onvansertib (25nM) reduced HPV- HNSCC spheroid growth more than HPV+ (n=2). Combining Plk1 inhibition with radiation also reduced colony formation (n=3, p&amp;lt;0.0001) and increased G2/M stalling (n=3) compared to either single agent in HPV- HNSCC cells. Functionally, onvansertib induces cleaved PARP and pPlk1 expression in HNSCC cells. However, pPlk1 is induced to a higher degree in HPV+ HNSCC cells potentially indicating a positive feedforward loop leading to decreased sensitivity to Plk1 inhibition. In HPV- but not HPV+ HNSCC cells, Plk1 expression is also increased after radiation emphasizing Plk1 as an attractive target to mitigate radioresistance in HPV- HNSCCs (n=3). Our findings that HPV+ HNSCC cells are more resistant to Plk1 inhibition than HPV- HNSCC cells provides novel mechanistic insight into these disease subtypes. As HPV- HNSCC is typically more resistant to standard therapies, these results support a novel combinatorial approach using an already approved Plk1 inhibitor with radiation to improve HPV- HNSCC patient outcomes. Citation Format: Julianna Korns, Samuel Thompson, Trisha Wise-Draper, Vinita Takiar. Plk1 signaling as a therapeutic target for HPV- head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2832.

Abstract 6720: Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors

Abstract H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (AT/RT) are aggressive pediatric brain tumors with dismal outcomes. DMG and AT/RT have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. This activation is manifested by elevated levels of ATF4 and NRF2 in DMG and AT/RT, respectively. Transient or low-level expression of these transcription factors protect cells from stress, while sustained, high-level activation leads to cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. We hypothesized that the high baseline level of ATF4 in DMG and NRF2 in AT/RT would make these tumors susceptible to ISR activators such as ixazomib. After determining the IC50 of ixazomib, DMG and AT/RT cell lines (JHH-DIPG1, JHH-DIPG16A, and CHLA06) were treated with increasing concentrations of the drug. Cleaved caspase 3 immunofluorescence showed a significant increase of apoptosis in all cell lines (JHH-DIPG16A and CHLA06 p&amp;lt;0.0001, JHH-DIPG1 p=0.0008 by ANOVA). Additionally, western blots for cleaved PARP and phospho-Rb expression detected induction of apoptosis and suppression of cell proliferation in JHH-DIPG1 treated with ixazomib. We are currently testing ixazomib in combination with idarubicin, a brain-penetrant anthracycline, and gemcitabine, a brain-penetrant nucleoside analog. Synergy testing in CHLA06 showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 with inhibitory concentrations of ixazomib and idarubicin in the low nanomolar range (scores of 10 or above indicate synergy). Similarly, ixazomib and gemcitabine synergized to suppress CHLA06 growth and induce apoptosis (67% Annexin V+ cells in combination compared to 14 and 24% Annexin V+ with single agent treatment). In the future, we will determine if ixazomib selectively kills DMG and AT/RT while sparing normal cells in iPSC brain organoids. We will also test ixazomib singly and in combination with traditional chemotherapy in orthotopic xenografts of AT/RT. These results suggest that ixazomib, especially in combination with idarubicin, gemcitabine or other ISR activators, has the potential to serve as an effective therapy for aggressive pediatric brain tumors. Citation Format: Orlandi Valencia Novak, Tyler Findlay, Jeffrey Rubens, Charles Eberhart, Eric H. Raabe. Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6720.

Abstract 4626: Spatial profiling of the tumor microenvironment in head and neck cancer identifies immune checkpoints and proteins involved in cell death signaling biomarkers of immunotherapy response

Abstract Immune checkpoint inhibitors (ICIs) Pembrolizumab and Nivolumab have shown promising results in patients with recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Nonetheless, only up to 20% of patients may benefit from them. Next generation predictive biomarkers of response to ICI therapies are currently needed. This retrospective study profiled pre-treatment tissue samples collected from R/M HNSCC patients (n=30) treated with Pembrolizumab or Nivolumab. Targeted spatial proteomics profiling was performed using NanoString’s GeoMx® Digital Spatial Profiler (GeoMx). To develop an informed region-of-interest (ROI) selection strategy for the GeoMx, we analyzed H&amp;E images from serial sections and performed Oncotopix® Discovery software, which considered pathology inputs and machine learning to analyze H&amp;E imagery and 4-channel GeoMx visualization inputs. We profiled 80 proteins simultaneously using the GeoMx across modules in cell death, immune activation, immune cell typing, immuno-oncology drug target, immune profiling, PI3K/AKT signaling, and pan-tumor markers.. Next, we performed differential expression (DE) analysis of the proteins localized in the tumor and stromal compartments against response to therapy parameters of complete, partial, stable and progressive disease (according to RECIST criteria). Four-channel GeoMx immunofluorescence images provided per-ROI cell counts (total and phenotyped based on 3 markers), and these data were used to aid in area and cell count normalization within the tumor and stromal compartments. We found that patients responsive to immunotherapy had higher expression levels of PD-L1, Bcl-2, BCLX, and BIM in the tumor, whereas VISTA, FOXP3, and CD66b were downregulated. In the stromal compartment, it was found that responders had higher expression levels of B7-H3, CD40, and SMA, but lower expression of PARP, S100B, and NY-ESO-1 in comparison to non-responders. In terms of best response analysis, patients with PR (n=5) had higher levels of PD-L1, ER-alpha, and CD68 expression, but lower levels of VISTA, CD27, and CD95/Fas in tumor regions than patients with PD (n=8). In the stroma, PD-L1, CD68, and HLA-DR were upregulated, while VISTA, BIM, and BAD were downregulated in patients with PR versus those with PD. We found that informed ROI selection strategy aided in defining key features in the tumor microenvironment for comparative analysis across samples, and for data normalization methods. Moreover, we found that immune checkpoints and proteins involved in cell death signaling play important roles in the immune responsive tumor microenvironment of HNSCC based on response to immunotherapy. Citation Format: Habib Sadeghirad, Ning Liu, James Monkman, Chin Wee Tan, Caroline Cooper, Jeni Caldera, Sarah Church, Fabian Schneider, James Robert Mansfield, Ken O'Byrne, Melissa Davis, Brett Hughes, Arutha Kulasinghe. Spatial profiling of the tumor microenvironment in head and neck cancer identifies immune checkpoints and proteins involved in cell death signaling biomarkers of immunotherapy response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4626.

Abstract 5610: [18F]FluorThanatrace ([18F]FTT) PET Imaging of PARP-inhibitor drug-target engagement as a biomarker of response in ovarian cancer

Abstract Purpose: Poly(ADP-ribose) polymerase enzyme inhibitors (PARPi) have become the standard-of-care treatment for homologous recombination deficient (HRD) high-grade serous ovarian cancer (HGSOC). However, not all HRD tumors respond to PARPi and biomarkers to predict response are needed. [18F]FluorThanatrace (FTT) is a PARPi-analog PET radiotracer that non-invasively measures PARP-1 expression. Herein, we evaluate the ability of FTT uptake to serve as a biomarker to predict response to PARPi in patient-derived xenograft (PDX) models and patients with HGSOC. Patients and Methods: In PDX models, FTT-PET was performed before and after PARPi (olaparib), ataxia-telangiectasia inhibitor (ATRi), or both. Changes in FTT were correlated with tumor size changes. Patients with HRD and HGSOC that were enrolled in CAPRI (PARPi+ATRi), LIGHT (PARPi only), or off-trial (PARPi only) were selected for this single-center, prospective, cohort, IRB-approved study. FTT-PET/CT imagining was obtained from the skull base to the proximal thighs on an Ingenuity TF scanner (Philips Healthcare) 60-90 minutes after intravenous infusion of 8-12 mCi FTT. Subjects were imaged with FTT-PET at baseline and after ~1 week of PARPi monotherapy treatment. Target lesions (primary tumor and/or metastases) were identified at the time of the baseline imaging on correlative anatomic imaging using RECIST 1.1 and maximum standardized uptake value (SUVmax) data, normalized by body weight, was collected. Changes in FTT-PET uptake were compared to changes in tumor size, CA-125, and progression free survival (PFS). Results: A decrease in FTT tumor uptake after 1 week of PARPi treatment correlated with response to PARPi+ATRi treatment in PARPi-resistant PDX models (r=0.81-0.83, P=0.1-0.22). In HGSOC patients (n=13), percent differences in FTT-PET after ~7 days of PARPi compared to baseline correlated with the first RECIST response (r=0.60, P=0.034), best RECIST response (r=0.75, P=0.01), best CA-125 response (r=0.73, P=0.033), and PFS (r=0.67, P=0.027). All patients with &amp;gt;50% reduction in FTT uptake had &amp;gt;6-month PFS and &amp;gt;50% reduction in CA-125 (P=0.004 and P=0.016, respectively). Utilizing only baseline FTT uptake correlated to best RECIST response (r=-0.65, P=0.035) but did not predict response when corelated with other measures. Importantly, a decrease in FTT uptake does not appear to be associated with a reduction in tumor burden or apoptosis in response to drug cytotoxic activity at this early timepoint, indicating specificity for drug-target engagement. Conclusions: The decline in FTT uptake shortly after PARPi initiation compared to baseline provides an in vivo measure of drug-target engagement and shows promise as an early biomarker to guide PARPi therapy. FTT-PET has both pre-clinical and clinical applications warranting further study, including guiding PARPi combination therapy. Citation Format: Sarah B. Gitto, Austin R. Pantel, Mehran Makvandi, Hyoung Kim, Sergey Medvedev, Joanna K. Weeks, Drew A. Torigian, Chia-Ju Hsieh, Benjamin Ferman, Nawar A. Latif, Janos L. Tanyi, Lainie P. Martin, Shannon M. Lanzo, Fang Liu, Quy Cao, Gordon B. Mills, Robert K. Doot, David A. Mankoff, Robert H. Mach, Lilie L. Lin, Fiona Simpkins. [18F]FluorThanatrace ([18F]FTT) PET Imaging of PARP-inhibitor drug-target engagement as a biomarker of response in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5610.

Antimutagenic 1,4-Dihydropyridine AV-153 Normalizes Expression of GLUT1, GLUT4, INOS, PARP1, and Gamma H2AX Histone in Myocardium of Rats with Streptozotocin Model of Diabetes Mellitus

Abstract Diabetic cardiomyopathy increases the risk of heart failure and worsens prognosis for diabetes mellitus (DM) patients. Its development depends on many factors, including modification of nitric oxide production and impaired DNA repair. The goal of the present work was to study in vivo effects of a 1,4-dihydropyridine AV-153, known as antimutagen and DNA-binder, on DNA integrity, and on the expression of several proteins involved in glucose transport, nitric oxide metabolism, and DNA repair in myocardium in diabetic rats. DM was induced in rats by streptozotocin (STZ) injection. Expression of proteins was studied by means of immunohistochemistry. Development of the STZ-induced DM significantly induced PARP1 and gamma H2AX histone, markers of DNA breakage, protein expression in heart tissue, while AV-153 administration decreased PARP1 and H2AX protein expression. In this model of diabetes, myocardial expression of iNOS was also significantly increased, but administration of AV-153 reduced it to normal levels. AV-153 also upregulated the expression of insulin-dependent GLUT4 and insulin-independent GLUT1 glucose transporters up to normal level in diabetic rats. Thus, AV-153 appears to be prospective for creation of a remedy for prevention of diabetic cardiomyopathy.

Analysis of the PARP1, ADP-Ribosylation, and TRIP12 Triad With Markers of Patient Outcome in Human Breast Cancer

PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.

Eriocitrin suppresses proliferation and migration of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPK pathway

To investigate the role of the ROS/MAPK signaling axis in mediating the inhibitory effect of eriocitrin on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells. SMMC-7721 cells were treated with different concentrations of eriocitrin for 24 h, and the changes in cell viability were detected with CCK-8 assay. The migration and invasion abilities of the treated cells were evaluated using Transwell and scratch healing assays, the cell proliferation was assessed with colony-forming assay, and changes in nuclear morphology were observed with DAPI staining. Western blotting was performed to examine the changes in the expressions of E-cadherin, N-cadherin, MMP-2, MMP-9, PARP, Pro-caspase 3, pJNK, p-P38, and p-ERK. The effect of eriocitrin on PARP cleavage in SMMC-7721 cells pretreated with ERK, JNK and P38 inhibitors (U0126, SB203580 and SP600125, respectively) was detected using Western blotting. The effect of treatment with Nacetyl-cysteine (NAC, 30 μmol/L) and eriocitrin (100, 200, and 300 μg/mL), alone or in combination, on reactive oxygen species (ROS) levels in the cells was examined using a DCFH-DA fluorescent probe. Eriocitrin below 50 μg/mL did not produce significant effect on the viability of SMMC-7721 cells (P>0.05). Treatment with eriocitrin significantly inhibited scratch healing, migration, and colony formation of the cells (P < 0.01), reduced the protein expressions of N-cadherin, MMP-2, and MMP-9 (P < 0.01), and up-regulated E-cadherin protein expression (P < 0.05). Eriocitrin-treated SMMC-7721 cells showed obvious apoptotic morphologies with decreased Procaspase 3 expression and increased PARP cleavage (P < 0.01) and phosphorylation levels of JNK, P38, and ERK (P < 0.01); Eriocitrin-induced PAPR cleavage was obviously enhanced by U0126 and SB203580 but attenuated by SP600125. Treatment with 300 μg/mL eriocitrin for 30 min significantly increased ROS level in the cells, and this effect was obviously suppressed by NAC. Eriocitrin can suppress the proliferation and migration and promote apoptosis of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPKs signaling pathway.

A Molecular Mechanism to Explain the Nickel-Induced Changes in Protamine-like Proteins and Their DNA Binding Affecting Sperm Chromatin in Mytilus galloprovincialis: An In Vitro Study.

Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present work, we analyzed PLs from Mytilus galloprovincialis by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl2 for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl2, while, at all NiCl2 doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl2, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl2. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed.

DNA damage and up-regulation of PARP-1 induced by columbin in vitro and in vivo

Columbin (CLB) is the most abundant (>1.0%) furan-containing diterpenoid lactone in herbal medicine Tinospora sagittate (Oliv.) Gagnep. The furano-terpenoid was found to be hepatotoxic, but the exact mechanisms remain unknown. The present study demonstrated that administration of CLB at 50 mg/kg induced hepatotoxicity, DNA damage and up-regulation of PARP-1 in vivo. Exposure to CLB (10 μM) induced GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1 and cell death in cultured mouse primary hepatocytes in vitro. Co-treatment of mouse primary hepatocytes with ketoconazole (10 μM) or glutathione ethyl ester (200 μM) attenuated the GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1, and cell death induced by CLB, while co-exposure to L-buthionine sulfoximine (BSO, 1000 μM) intensified such adverse effects resulting from CLB exposure. These results suggest that the metabolic activation of CLB by CYP3A resulted in the depletion of GSH and increase of ROS formation. The resultant over-production of ROS subsequently disrupted the DNA integrity and up-regulated the expression of PARP-1 in response to DNA damage, and ROS-induced DNA damage was involved in the hepatotoxicity of CLB.